Migraine is one of the most common neurological disorders and a big burden of health system. Current treatments for migraine headache often fall short. The serotonin 1D receptor (5-HT1DR) on the central terminals of the trigeminal ganglion (TG) neurons is the prime target of the triptan family of anti-migraine drugs. Triptans bind to 5-HT1DR and activate multiple Gi/o-coupled signal transduction pathways, mainly the inhibition of voltage-gated Ca2+ channel (VGCC) activity and the subsequent reduction of neurotransmitters/neuropeptides release from TG terminals. One of the strategies to develop new migraine therapeutics is to selectively enhance the synthesis, plasma membrane trafficking and/or intracellular signaling of 5-HT1DR in TG neurons. Proteins that interact with the third intracellular loop of human 5-HT1DR has been identified through yeast two- hybrid screening. PLEKHB1, a pleckstrin homology (PH) domain-containing integral membrane protein, preferentially interacts with 5-HT1DR but not the other 11 subtypes of 5-HT receptors. The expression of endogenous PLEKHB1 and 5-HT1DR proteins overlaps substantially in TG neurons. Furthermore, the interaction between endogenous PLEKHB1 and 5-HT1DR in TG tissue is confirmed by co-immunoprecipitation experiment. Over-expression of PLEKHB1 decreases the expression of 5-HT1DR in HEK cells as well as cultured TG neurons. Importantly, PLEKHB1 significantly reduces 5-HT1DR-mediated inhibition of VGCC currents in HEK cells. The research objective of this proposal is to test the hypothesis that PLEKHB1 modulates the metabolism and signaling of 5-HT1DR, thereby regulating the activity of TG neurons as well as the gain of the neuronal circuit underlying migraine headache. First, we will investigate whether PLEKHB1 modulates 5-HT1DR protein level and plasma membrane expression in TG neurons. Second, we will test if PLEKHB1 modulates signaling through 5-HT1DR in TG neurons, focusing on the effects of PLEKHB1 on 5-HT1DR-mediated inhibition of VGCC currents as well as synaptic transmission at TG terminals. Finally, we will investigate whether PLEKHB1 regulates the nocifensive behavior as well as the activity-dependent gene expression in a mouse model of headache. In particular, we will test if PLEKHB1 affects the efficacy and/or potency of triptans in the mouse models of headache. Together, the outcome of this project will not only offer valuable insights into the functional significance of PLEKHB1 in headache generation and modulation, but also lay important ground work for the development of mechanism-based screening of new migraine therapeutics.